Lentiviral triplex dna, and vectors and recombinant cells containing lentiviral triplex dna

ABSTRACT

The present invention provides nucleic acid, vectors, viruses, and recombinant cells comprising triple-stranded structures, such as those resulting from central initiation and termination of HIV-1 reverse transcription at the center of HIV-1 linear DNA genomes. These triplex structures can act as a cis-determinant of HIV-1 DNA nuclear import, allowing infection of non-dividing target cells. In one aspect, the presence of the DNA triplex sequence in an HIV vector strongly stimulates gene transfer in hematopoietic stem cells. The invention also provides methods of using these triplex structures for making recombinant cells, as well as methods of using the recombinant cells to express proteins of interest both in vitro and in vivo.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.13/942,180, filed Jul. 15, 2013, which is a continuation of U.S.application Ser. No. 13/301,147, filed Nov. 21, 2011, now U.S. Pat. No.8,512,993, which is a continuation of U.S. application Ser. No.11/291,390, filed Dec. 1, 2005, now U.S. Pat. No. 8,093,042, which is acontinuation of U.S. application Ser. No. 09/685,343, filed Oct. 11,2000, now abandoned. U.S. application Ser. No. 09/685,343, which dependson, and claims the benefit of the filing date of, U.S. ProvisionalApplication Ser. No. 60/158,387, filed Oct. 12, 1999, the entiredisclosure of which is hereby incorporated by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to the field of biotechnology, andespecially to viral nucleic acids and cells containing viral nucleicacids. More particularly, the present invention relates to viral nucleicacid sequences that can be part of triplex DNA structures as well asnucleic acid vectors, viruses, and cells containing these viral nucleicacid sequences. It further relates to methods of using such DNAstructures and nucleic acid sequences.

2. Description of Related Art

Gene transfer in hematopoietic stem cells (HSC) has great potential,both for gene therapy of inherited as well as acquired diseases, and forthe understanding of mechanisms regulating normal and pathologicalhematopoiesis. As HSC have extensive proliferative capacities, stablegene transfer should include genomic integration of the transgene.Retroviruses based on Moloney murine leukemia virus (MoMLV) have beenvery popular because they integrate into the host cell genomes and canallow long-term transgene expression. However, these oncoretroviruses donot integrate in non-dividing cells, and most HSC are quiescent. Manypre-stimulation protocols using different cytokine associations havebeen developed to trigger cycling of HSC in order to render themtransducible by oncovirus-derived, mitosis-dependant gene transfervectors. However, cytokine stimulation induces differentiation togetherwith proliferation and potentialities of HSC could be lost during thetransduction process. This problem can be overcome by usinglentivirus-derived vectors since lentiviruses have been shown to infectboth dividing and non-dividing cells (Poznansky et al., 1991; Naldini etal., 1996). Initial reports published in the last three years using suchlentivirus-derived vectors for transduction of HSC showed promising butvery heterogenous results (Case et al., 1999; Evans et al., 1999; Uchidaet al., 1998; Miyoshi et al., 1999).

Lentiviruses have evolved a nuclear import strategy which allows theirDNA genome to cross the nuclear membrane of a host cell. This activenuclear import of lentiviruses accounts for their unique capacity, amongretroviruses, to replicate efficiently in non-dividing target cells,such as tissue macrophages. The restriction of replication ofoncoviruses like MoMLV to dividing cells appears to be due to therequirement for disruption of the nuclear membrane barrier duringmitosis, allowing MoMLV pre-integration complexes (PICs) to enter thenucleus (Roe et al., 1993). Mitosis-independent replication oflentiviruses, at the origin of their in vivo replication strategy andhence of their pathogenicity, has also enabled the generation oflentiviral gene transfer vectors with promising therapeutic applications(Poznansky et al., 1991; Naldini et al., 1996).

The mitosis-independent replication of lentiviruses was firstdemonstrated by the productive infection of non-mitotic chondroid cellsby the VISNA lentivirus (Thormar, 1963). Soon after its discovery, HIVwas shown to replicate in differentiated primary macrophages (Gartner etal., 1986; Ho et al., 1986). HIV DNA integrates in the chromatin ofnon-mitotic target cells (Weinberg et al., 1991; Lewis et al., 1992),implying that HIV-1 PICs are able to cross the nuclear membrane of hostcells (Bukrinsky et al., 1992). Thus, mitosis-independent nuclear importis a pivotal event responsible for the ability of lentiviruses toreplicate in non-dividing cells.

The search for the viral determinants responsible for the active nuclearimport of the HIV1 DNA genome has constituted an active butcontroversial field of investigation. The presence of putative nuclearlocalization signals (NLSs) within the matrix (MA) and Vpr viralproteins has led to the proposition that they could act in a redundantmanner in HIV-1 DNA nuclear import (Bukrinsky et al., 1993b; Emerman etal., 1994; Popov et al., 1998; von Schwedler et al., 1994). It has beenproposed that phosphorylation of a small subset (1%) of MA molecules ata C-terminal tyrosine residue triggers their release from the plasmamembrane and their association with HIV-1 integrase protein (Gallay etal., 1995a; 1995b). The contribution of these proteins to thekaryophilic properties of HIV-1 PICs is currently a matter of strongdebate (Freed and Martin, 1994; Freed et al., 1995; Fouchier et al.,1997; Freed et al., 1997; Koostra and Schuitemaker, 1999). Morerecently, NLS motifs have been identified in the integrase protein (IN)and mutations in these motifs have been reported to abolish theinteraction of IN with karyopherin a, a cellular NLS receptor (Gallay etal., 1997).

SUMMARY OF THE INVENTION

Whatever the role of these candidate viral proteins in HIV nuclearimport, the present invention shows that the retrotranscribed HIV-1genome itself bears a cis-acting determinant for its nuclear import.

The invention provides a nucleic acid comprising a triple-stranded(triplex) structure, such as one from a lentivirus. The triplexstimulates entry of nucleic acids into the nucleus of cells. The nucleicacid can contain the cPPT and CTS cis-acting sequences of a lentivirus.The lentivirus can be any lentivirus, including, but not limited to,HIV-1, HIV-2, VISNA, EINV, FIV, and CAE. In embodiments, the nucleicacid is in the context of a vector, such as an expression vector.

Thus, the invention also provides a vector, for example, a nucleic acidvector. The nucleic acid vector can include sequences from any vectorknown to the skilled artisan as useful for transfer of nucleic acidsinto cells or for expression of nucleic acids in vivo or in vitro.

The invention further provides viruses and cells (eukaryotic andprokaryotic) containing the nucleic acid of the invention. The cells canbe recombinant cells.

The invention additionally provides a method of transferring nucleicacid to a host cell nucleus by, for example, exposing the host cell tothe nucleic acid, vector, virus, or cell of the invention, to provide arecombinant cell. The method of the invention permits high-efficiencytransfer of nucleic acids to the host cell nucleus, such as the nucleusof a hematopoietic stem cell. High-efficiency transfer permits theskilled artisan to practice various methods of treatment, including, butnot limited to, methods of prophylactic treatment, methods ofameliorative treatment, and methods of curative treatment. For example,methods of gene therapy are enabled by this invention. In general, genetherapy can be used to treat blood diseases, brain diseases, viraldisease, as well as many other inherited and acquired diseases.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A-C shows that central initiation of reverse transcription is animportant step in the replication cycle of HIV-1.

-   (A) Mutations introduced in the HIV-1 cPPT sequence. Conservative    and semiconservative cPPT mutant viruses were constructed.    Semi-conservative mutant cPPT-D contains 10 mutations in the 19-mer    cPPT. Mutant cPPT-AG is its control virus in which a single purine    to purine mutation introduces the same amino acid change in the    overlapping integrase coding region. Mutations are shown in reversed    type.-   (B) Impact of the mutations in the cPPT on HIV-1 infectivity. Virus    replication kinetics on PHA stimulated PBLs (left panel) and MT4    cells (right panel). Cells were infected with equivalent amounts of    viral particles according to the capsid antigen (p24) contents of    viral supernatants. Virus production was followed over time by RT    activity.-   (C) Single cycle virus titrations in dividing or non-dividing    aphidicolin-treated P4 cells (HeLa CD4-LTR LacZ).-galactosidase    activity was measured using a chemiluminescent assay. Results are    expressed as relative light units (RLU)/sec/ng p24 of the inoculum,    mean SD of four independent experiments.

FIG. 2A-C depicts mutations in the cPPT that do not affect virusproduction, viral DNA synthesis nor the ability of viral DNA tointegrate in vitro.

-   (A) Effect of mutations in the cPPT on virus production. HeLa cells    were transiently transfected with pLAI, pcPPT-AG, or pcPPT-D    plasmids. Virus production was measured by quantitation of p24 viral    antigen in cell supernatants, 48 hours post-transfection.-   (B) Effect of mutations in the cPPT on reverse transcription    efficiency. P4 cells were infected with the same amounts of viral    particles (300 ng of p24 antigen per 106 cells) and DNA was    extracted 12 hours later. Total amounts of reverse transcribed viral    DNA, represented by an internal Mscl HIV-1 fragment, was detected by    Southern blot using the same DNA fragment as a probe and quantitated    using a phosphorimager.-   (C) Effect of mutations in the cPPT on in vitro integration. MT4    cells were co-cultivated with H9-LAI or H9-cPPT-225 chronically    infected cells. In vitro integration of viral preintegration    complexes, isolated from the cytoplasm of infected cells, was    performed as previously described (Farnet and Haseltine, 1990). Each    lane is loaded with cytoplasmic DNA from 2×10⁸ infected cells.

FIG. 3A-C shows that central DNA triplex mutant viruses are defective innuclear import of viral DNA.

-   (A) Strategy for the quantitative follow up of the synthesis,    circularization, and integration of HIV-1 DNA. DNA from infected    cells was extracted at various times postinfection, digested with    Mscl and Xhol, and analyzed by Southern blot using a probe    overlapping the 5′Mscl site. The internal 1.9 kb DNA fragment,    common to all viral DNA species irrespective of their integrated or    unintegrated state, indicates the total amount of viral DNA in    infected cells. The 2.6 kb, 2.8 kb, and 3.4 kb signals    corresponding, respectively, to unintegrated linear DNA, one, and    two LTRs circular DNAs are revealed. Since the PCR generated probe    exactly overlaps the Mscl site, the intensity of each band is    directly proportional to the amount of the corresponding viral DNA    species. Thus, the amount of integrated proviral DNA can be    calculated by subtracting from the total amount of viral DNA the    signals of unintegrated linear and circular viral DNA.-   (B) Southern blot analysis of viral DNA processing in infected    cells. P4 cells were infected with equivalent amounts of each virus,    normalized on the p24 contents of the supernatants. Infected cells    were lysed at different times post-infection, DNA was extracted, and    used for the quantitative analysis described above.-   (C) Intracellular viral DNA profiles, on completion of one cycle of    infection (48 hours post-infection). Results are expressed as    percentages of total viral DNA. Similar intracellular viral DNA    profiles were obtained using MT4 cells (data not shown).

FIG. 4A-B shows that linear DNA from central DNA triplex mutant virusesaccumulates at the vicinity of the nuclear membrane.

-   (A) Nucleus/cytoplasm fractionation of infected P4 cells. Southern    blot analysis of viral DNA from nuclear (N) and cytoplasmic (C)    fractions, 24 hours post infection. Fractionation based on triton    lysis was performed as previously described (Belgrader et al.,    1991). DNA was restricted with Mscl and hybridized using the Mscl    site overlapping probe.-   (B) Detection of individual HIV-1 genomes by Fluorescence In Situ    Hybridization (FISH). P4 cells were infected at high multiplicity (2    g of p24 antigen per 106 cells), and hybridized using a full length    HIV-1 genome probe. Fluorescent signals were amplified by tyramid    precipitation. Optical sections through cells were analyzed by    deconvolution microscopy.

FIG. 5A-D shows that the insertion of the central DNA triplex in anHIV-1 based vector enhances GFP transduction and nuclear import of thevector DNA genome.

-   (A) Schematic diagram of the vector genomes. cPPT and CTS central    cis active sequences of the HIV-1 genome, responsible for the    formation of the DNA triplex during reverse transcription, were    inserted in a central position in the previously described HR GFP    vector (Naldini et al., 1996). TRIPinv-GFP includes the central DNA    triplex sequence in the reverse, non-functional orientation.-   (B) Comparative efficiency of GFP transduction using HIV vectors    with or without a central DNA triplex. Dividing or non dividing    (aphidicolin treated) HeLa cells were used as targets. GFP    fluorescence was quantitated 48 hours post-transduction using a    microplate fluorometer (Wallac). Results are expressed as the mean    SD of a representative experiment performed in triplicate.    Pseudotransduction of GFP activity was subtracted from the signal.-   (C) Southern blot analysis of vector DNA processing in transduced    cells. Transduced HeLa cells were lysed at different times    post-infection, DNA was extracted, restricted, and Southern blotted    using a similar strategy as for the viruses. Mscl digestion is    replaced by EcoNI and AvaH, and the probe is a PCR DNA fragment    overlapping exactly the EcoNI site.-   (D) Quantitative analysis of vector DNA intracellular status, 48    hours post infection. Results are presented as percentages of total    vector DNA.

FIG. 6A-B depicts a model for DNA triplex dependent HIV-1 genome nuclearimport.

-   (A) Overview of the observed phenotype of central DNA triplex mutant    viruses. Central initiation and termination steps of HIV-1 reverse    transcription creates a long plus strand DNA flap structure: the    central DNA triplex. HIV-1 plus strand is synthesized as two    discrete halfgenomic segments. A downstream segment is initiated at    a central copy of the polypurine tract sequence (cPPT). The upstream    segment terminates downstream the cPPT, after a 99 nucleotide long    strand displacement event, blocked by the central termination    sequence (CTS). At completion of a single cycle of infection, viral    DNA from wild-type virus is almost fully processed into integrated    provirus (55%), 1 LTR (35%), and 2LTRs circular DNA (<5%), while a    small fraction remains as linear DNA (<10%). Mutations in the cPPT    affects the formation of the central DNA triplex. The final reverse    transcription product of a central initiation mutant virus is a    continuous double-stranded linear DNA lacking the central DNA    triplex (Chameau et al., 1992). Viral DNA from cPPT-D mutant virus    accumulates in infected cells as linear DNA molecules, and localizes    at the vicinity of the nuclear membrane.-   (B) Two speculative mechanisms for triplex dependent HIV-1 genome    nuclear import via maturation of the reverse transcription complex    (RTC) into a pre-integration complex (PIC) and linear DNA    translocation through the nuclear pore. HIV-1 reverse transcription    probably occurs within a structured (ordered) complex surrounded by    an assembly of capsid proteins. The size of RTC exceeds the    exclusion diameter of the nuclear pore. Before translocation, RTC    undergoes a maturation into a smaller PIC with loss of the capsid    proteins (Karageorgos et al., 1993). Formation of the DNA triplex    signals the end of the viral DNA synthesis and could signal the    escape of the HIV DNA from the capsid assembly. In the PIC, the    extremities of the linear DNA are probably bridged together after    dimerization of integrase proteins bound to the tips of the LTRs    (Miller et al., 1997). The DNA triplex being at a central position,    one logical structure for HIV-1 PIC would be a double filament,    symmetrically folded on either side of the central triplex by the    integrase dimerization. The DNA triplex would then constitute an    apex which could interact with karyophilic shuttling proteins,    allowing the passage of the HIV-1 DNA filament through the nuclear    pore. In cPPT mutant viruses, a default of RTC maturation into PIC    would induce an accumulation of integral viral capsids at the    nuclear pore. Alternatively, the absence of the DNA triplex in cPPT    mutant linear DNA would prohibit the interaction with shuttling    proteins forbidding translocation of HIV-1 genome through the    nuclear pore. In both cases, DNA from cPPT mutant viruses    accumulates as linear DNA at the vicinity of the nuclear membrane.

FIG. 7A-B shows the results of transduction experiments using CD34+human cord blood cells.

-   (A) FACS analysis of human cord blood CD34+ cells transduced for 24    hours in the presence of 100 ng/ml of viral P24 in conditions    described herein. Analysis was performed 48 hours after washing of    the cells at the end of the 24 hour transduction period. Percentages    are expressed as proportions of morphologically gated hematopoietic    cells. X mean indicates the mean value of green fluorescence    intensity.-   (B) GFP expression was analyzed at day 5 post transduction.

FIG. 8A-C diagrammatically represents the effect of viral dosage ontransfection efficiency.

-   (A) The percentage of CD34+ cells expressing eGFP.-   (B) The mean value of GFP fluorescence intensity of CD34+eGFP+    cells. (C) The mean value of GFP fluorescence intensity of    CD34+eGFP+ cells multiplied by the percentage of CD34+eGFP+ cells.    CD34+ cells were transduced for 24 hours under the conditions    specified herein with the lentiviral vector including the eGFP    coding sequence under the control of the CMV promoter, and including    (thick line) or without (dashed line) the triplex structure.    Analysis was performed 48 hours after washing of the cells at the    end of the 24 hour transduction period.

FIG. 9A-B depicts FACS analysis of human cord blood CD34+ cellstransduced for 24 hours under the conditions described in the text witha lentiviral vector having an intact HIV-1 LTR (left panels) or a U3deleted HIV-1 LTR (right panels) and an internal CMV promoter (upperpanels) or the EF-1 alpha promoter (lower panels).

-   (A) Analysis was performed 48 hours after washing of the cells at    the end of the 24 hour transduction period.-   (B) Analysis was performed 120 hours after washing of the cells at    the end of the 24 hour transduction period. The analysis    distinguishes between bright (immature) and dull CD34 cells.

Percentages are expressed as proportions of morphologically gatedhematopoietic cells. The second number, when indicated, represents themean of green fluorescence.

DETAILED DESCRIPTION OF THE INVENTION

We have previously shown that HIV-1 has evolved a complex reversetranscription strategy, which differs from that of oncoviruses by twosteps occurring at the center of the genome: one additional initiationof plus strand synthesis coupled with a termination step. HIV1 plusstrand DNA is synthesized as two discrete half-genomic segments. Anadditional copy of the polypurine tract cis-active sequence, present atthe center of all lentiviral genomes (cPPT), initiates synthesis of adownstream plus strand (Chameau et al., 1991,1992). The upstream plusstrand segment initiated at the 3′PPT will, after a strand transfer,proceed to the center of the genome and terminate after a discretestrand displacement event (FIG. 6A). This last chronological event ofHIV-1 reverse transcription is controlled by the central terminationsequence (CTS) cis-active sequence, which ejects HIV-1 reversetranscriptase (RT) at this site in the specific context of stranddisplacement synthesis (Chameau et al., 1994; Lavigne et al., 1997).Thus, the final product of HIV-1 reverse transcription is a linear DNAmolecule bearing in its center a stable 99 nucleotide long DNA flap,here referred to as the central DNA triplex (FIG. 6A).

Central initiation and termination mutant viruses both synthesize areverse transcription product devoid of a wild type DNA triplex. Asdepicted in FIG. 6A, in the case of a central initiation mutant, thefinal product is a continuous double-stranded linear DNA lacking thecentral triplex (Chameau et al., 1992). The downstream plus strandsegment initiated at the cPPT is not synthesized. Thus, no stranddisplacement occurs at the center of the genome; elongation of thetransferred plus strand proceeds all along the genome. In the case of acentral termination mutant, central strand displacement events are nolonger controlled by the mutated CTS sequence and longer, randomlydistributed plus strand overlaps are generated, as compared to thediscrete wild type DNA triplex (Chameau et al., 1994). Mutations in thecPPT or CTS cis-active sequences severely impair HIV replication,suggesting a direct role of the central triplex in the retroviral lifecycle (Chameau et al., 1992; Hungnes et al., 1992; Chameau et al.,1994).

The present invention discloses that the central DNA triplex of HIV-1 isinvolved in a late step of HIV-1 genome nuclear import, immediatelyprior to, or during, viral DNA translocation through the nuclear pore.Hence the distinctive features of lentiviral reverse transcriptionaccount, at least in part, for the unique capacity of lentiviruses,among retroviruses, to replicate in non-dividing cells. The inventionalso discloses that HIV gene transfer vectors lacking the central DNAtriplex exhibit a strong nuclear import defect. This inventionestablishes that the insertion of the central cis-active sequences ofthe HIV-1 genome into a previously described HIV vector (Naldini et al.,1996) increases the transduction efficiency by complementing the nuclearimport defect of the original vector DNA to wild type levels. Thisfinding provides additional and independent evidence for the role of thecentral DNA triplex in HIV-1 nuclear import. The description of the DNAtriplex as a nuclear import determinant of lentiviruses has importantimplications for the design of efficient lentiviral vectors. Since theinfection of non-dividing target cells by lentiviruses relies on theiruse of an active nuclear import pathway, it is preferable to maintainthe lentiviral nuclear import determinants in derived vector constructs.Classical retroviral vector constructs are replacement vectors in whichthe entire viral coding sequences between the LTRs are deleted andreplaced by the sequences of interest (Miller A D, 1997). In the case oflentiviral vectors, this classical strategy leads to the deletion of thecentral cis-active sequences cPPT and CTS. The important role of thetriplex in HIV nuclear import implies that such replacement vectorconstructs are not optimal. This finding establishes the fact that theDNA triplex nuclear import determinant is operative in the heterologouscontext of an HIV-1 derived lentiviral vector. The DNA triplex per seout of the context of the native HIV-1 genome can promote nuclear importof heterologous DNA sequences (PCT France No. 98 05197, 24 Apr. 1998).The presence of the DNA triplex sequence induces a marked increase ofgene transduction efficiency in hematopoietic stem cells.

Thus, in one aspect, the present invention provides nucleic acids (DNAor RNA, or analogs thereof) that are capable of participating in triplexnucleic acid structures (i. e., triple-stranded nucleic acids). Inembodiments of the invention, the nucleic acids are lentiviralsequences. In embodiments, the nucleic acids are derived from lentiviralsequences, such as by directed mutagenesis (i. e., intentional deletion,insertion, or replacement of at least one nucleotide) or selectivepressure mutagenesis. The nucleic acids of the invention permithigh-efficiency transfer of nucleic acids into host cells, andespecially into host cell nuclei. Because of this ability, genes thatare operably or physically linked to the nucleic acids of the inventioncan be expressed at high levels and/or in a high percentage of cellsexposed to the nucleic acids. DNA inserted in a triple-stranded regionof a lentiviral genome in accordance with the present invention isparticularly stable in the construct, and aids in achieving a high levelof transduction and transfection.

In a preferred embodiment, the invention provides a three-stranded DNAsequence induced by the cPPT and CTS regions of a lentivirus, which iscapable of inducing a high rate of entry of vector DNA into a host cellnucleus, or capable of increasing the rate of nuclear import of vectorDNA. In embodiments, the three-stranded DNA sequence can be covalentlylinked to a heterologous nucleic acid sequence, such as a reporter geneor gene of other interest. For example, the nucleic acid of theinvention can comprise a heterologous nucleic acid sequence that encodesa peptide, polypeptide, or protein. In embodiments, the heterologousnucleic acid sequence is present within the triple-helix region. Inembodiments, the heterologous nucleic acid sequence is present on thesame nucleic acid as the triple-helix region, but outside of the triplehelix. In embodiments, a single nucleic acid comprises more than onethree-stranded sequence induced by the cPPT and CTS regions of alentivirus.

In another aspect, the present invention provides vectors, such asshuttle vectors, expression vectors, integration vectors, transposons,retrotransposons, and the like, which contain at least one sequencecapable of participating in the formation of triplex nucleic acidstructures. In embodiments, the vector comprises a single sequencecapable of participating in the formation of triplex nucleic acidstructures. In embodiments, the vector comprises more than one sequencecapable of participating in the formation of triplex nucleic acidstructures. The vectors can be used to transfer the nucleic acids of theinvention from one cell to another, to aid in generation of largequantities of the nucleic acids of the invention, and to serve as a basemolecule into which other nucleic acid sequences of interest (e. g.,reporter genes, therapeutic genes) can be inserted.

In yet another aspect, the present invention provides a method forefficiently infecting, transfecting, or transducing cells with a viralnucleic acid sequence comprising at least one sequence that canparticipate in triplex nucleic acid structures. In embodiments, themethod comprises exposing a cell, or a plurality of cells, to at leastone copy of a nucleic acid, vector, virus, or cell of the invention. Inembodiments, the step of exposing the cell or cells to the nucleic acid,vector, virus, or cell of the invention is conducted under conditionssuch that the nucleic acid, vector, virus, or cell of the invention canenter the target (host) cell or cells. In preferred embodiments, theconditions are such that high levels of the nucleic acid, vector, virus,or cell of the invention enter the target cell or cells.

In embodiments, the method is highly efficient, permitting insertion ofthe nucleic acid of the invention into the nucleus of 30% or greater ofthe cells exposed to the nucleic acid. In embodiments, the percentage ofnuclei taking up the nucleic acid of the invention is 40% or greater,for example 50% or greater, 60% or greater, 70% or greater, 80% orgreater, or 90%-100%. In preferred embodiments, the percentage of nucleitaking up the nucleic acid of the invention is greater than 80%, morepreferably, greater than 85%, and most preferably, greater than 90%,such as greater than 95%. In embodiments, the cells are infected ortransfected with whole viruses (including phages) or bacteria containingthe nucleic acids of the invention. In embodiments, the nucleic acids ofthe invention are introduced into the cells using mechanical and/orchemical means (e. g., electroporation, liposome fusion). Inembodiments, naked nucleic acid according to the invention is taken upby target host cells.

Thus, in an embodiment, the invention provides the use of a nucleotidesequence comprising the cPPT and CTS regions, which adopts athree-stranded DNA structure (triplex) after reverse transcription, in alentiviral or retrotransposon vector and which stimulates entry of, andthe rate of nuclear import of, the vector DNA into the nucleus of atransduced cell.

In an aspect of the invention, recombinant cells are provided thatcontain the nucleic acids or vectors of the invention. The recombinantcells can be any cell (prokaryotic or eukaryotic), including a progenyor derivative thereof. Thus, the invention includes cells that arecreated using a cell of the invention. In embodiments, the cells areeukaryotic. In embodiments, the cells are mammalian cells, such as HeLacells or hematopoietic cells, such as hematopoietic stem cells. Thus, inembodiments, the invention provides a process of transducing eukaryoticcells, wherein the process comprises use of a three-stranded DNAsequence induced by the cPPT and CTS regions of a lentivirus, which iscapable of inducing a high rate of entry of vector DNA into a host cellnucleus, or capable of increasing the rate of nuclear import of vectorDNA.

Because the recombinant cells of the invention can express aheterologous gene of interest at high levels, these cells can be used innumerous applications. Applications include, but are not limited to,production of high levels of proteins of interest (such as proteins oftherapeutic value) in cell culture and production of a protein ofinterest in vivo by introduction of the recombinant cell of theinvention into an individual in need of the protein. The individual canbe an animal or a human. Accordingly, the invention has bothveterinarian applications as well as medical ones.

In embodiments, this aspect of the invention provides a method ofproducing a recombinant protein of interest by exposing a target cell toa nucleic acid, vector, or virus of the invention and permitting thetarget cell to take up nucleic acid of the invention, for example,through transduction, transformation, or transfection. Followingintroduction of the nucleic acid into the target cell, the cell iscultured under conditions whereby the recombinant protein of interest isexpressed, and thus produced by the target cell, which now is considereda recombinant cell according to the invention. Although the method canbe practiced on a single, individual cell, it is evident that the methodwill often be more practical if practiced on a collection of cells inwhich all of the cells are clones. As used herein, “cell” refers to anindividual cell or a collection of identical cells.

The method can further include purifying or isolating the protein ofinterest from the recombinant cell or cell culture fluid. In such amethod, protein expression and purification procedures known to those ofskill in the art can be applied. These procedures are well known tothose of skill in the art and therefore need not be detailed here.

In a preferred embodiment, this aspect of the invention provides aprocess for expressing a gene of interest in vitro, wherein the processcomprises: a) exposing target cells to an isolated or purified nucleicacid comprising a gene of interest and at least one copy of the cPPT andCTS cis-acting regions of a retrovirus, wherein the cPPT and CTS regionsinduce a three-stranded DNA structure, under conditions that permituptake of the nucleic acid into the target cell to create a recombinantcell, and b) culturing the recombinant cell under conditions that permitat least part of the nucleic acid to be transferred to the nucleus ofthe recombinant cell and the gene of interest to be expressed. Inembodiments, the process uses a vector according to the invention.

Thus, the invention provides a method of expressing a gene of interestin vitro, for example, in tissue culture. In embodiments, the methodcomprises exposing target cells to a nucleic acid, vector, virus, orcell of the invention under conditions where the target cell can take upthe molecule of the invention containing the gene of interest. Therecombinant cell thus made is then allowed to grow and replicate underconditions where the gene of interest is expressed. In embodiments, thein vitro method of gene expression is coupled to a method of purifyingor isolating the protein of interest. In these embodiments, the proteinof interest can be purified or isolated from other cellular componentsusing techniques known to those of skill in the art, including, but notlimited to, liquid chromatography, precipitation, centrifugation, gelfiltration, and affinity chromatography. Suitable techniques are knownto those of skill in the art and need not be detailed here.

Thus, the invention also provides a method of expressing a gene ofinterest in vivo, for example, in an individual in need of the proteinexpressed by the gene. In embodiments, the method of expressing a genein vivo comprises making a recombinant cell outside the individual byexposing a host cell to a nucleic acid, vector, virus, or cell of theinvention, to make a recombinant cell according to the invention. Therecombinant cell of the invention is then administered to, introducedinto, or otherwise exposed to, the individual, whereupon the gene ofinterest is expressed. For example, the method can compriseadministering a recombinant cell comprising a nucleic acid of theinvention to an individual, and permitting the recombinant cell toexpress the nucleic acid within the individual's body. In preferredembodiments, the recombinant cell is a hematopoeitic stem cell. Inembodiments, the recombinant cells are first purified or isolated fromnon-recombinant cells, then administered to, introduced into, orotherwise exposed to, the individual.

In other embodiments, the method of expressing a gene in vivo comprisesexposing (e. g., administering, introducing, etc.) the individual to anucleic acid, vector, and/or virus of the invention. In embodiments, thenucleic acid, vector, and/or virus transfects/transduces/infects atleast one of the individual's cells, whereupon the gene of interest isexpressed. For example, the method can comprise administering a nucleicacid, vector, or virus of the invention to an individual in an amountand form sufficient to result in expression of the gene of interestwithin the individual's body. Preferably, the method results inexpression of the gene of interest in a target tissue or cell.

In an embodiment, this aspect of the invention provides a process fortreating an individual suffering from, or having a high likelihood ofdeveloping, a disease or disorder having a genetic basis. The processcomprises administering a retroviral vector comprising a) a nucleic acidencoding a therapeutic protein and b) at least one copy of the cPPT andCTS cis-acting regions of a retrovirus, wherein the cPPT and CTS regionsinduce a three-stranded DNA structure, to the individual in an amountsufficient to result in expression of the therapeutic protein in anamount sufficient to treat the disease or disorder. The treatment can beprophylactic, ameliorative, or curative. The process can treat a blooddisease or disorder, a brain or nervous system disease or disorder, or adevelopmental disease or disorder. Techniques for introducing and/orexpressing genes in vivo are known to those of skill in the art. Thepractitioner may select the technique most suitable for the givenprotein or target tissue or cell.

Accordingly, the invention provides a process of treating a hostcomprising use of a retroviral vector containing a three-stranded DNAsequence induced by the cPPT and CTS regions of a lentivirus, which iscapable of inducing a high rate of entry of vector DNA into a host cellnucleus, or capable of increasing the rate of nuclear import of vectorDNA.

In accordance with the above aspects of the invention, a kit is alsoprovided. The kit can contain at least one nucleic acid, at least onevector, at least one virus, or at least one cell of the invention, or acombination of any or all of those. The kit can provide each of theabove embodiments of the invention together in a single composition orseparately, as for example, in different containers. In embodiments, thekit includes some or all of the reagents and supplies necessary to usethe nucleic acids, vectors, viruses, and cells of the invention for thedesired purpose.

The present invention discloses an original mechanism of HIV-1 nuclearimport with a crucial role of a three stranded DNA structure, the DNAtriplex, in this mechanism. HIV-1 has evolved a complex reversetranscription strategy, whereby a central strand displacement event,consecutive to the central initiation and termination of reversetranscription, creates a DNA triplex at the center of unintegratedlinear HIV-1 DNA molecules. This DNA triplex acts in turn as acis-active determinant of the nuclear import of the HIV-1 genome. Theinvention shows that central initiation and termination, two distinctivesteps of HIV-1 reverse transcription, account for the capacity of HIV-1to infect non-dividing target cells.

The Examples of the invention further show that lack of the DNA triplexleads to a virus which is almost non-infectious in dividing ornon-dividing cells. Although mutations in cPPT do not affect the rate ofsynthesis of viral DNA or its ability to integrate in vitro, most of theretrotranscribed DNA molecules from the cPPT mutant virus accumulateover time as unintegrated linear DNA. In contrast, linear DNA from thewild-type virus is almost fully processed into integrated proviruses andDNA circles. The intracellular DNA profile of cPPT mutant viruses pointsto a defect of nuclear import, the viral DNA accumulating as linearmolecules as a consequence of its lack of access to the nuclearcompartment where it could integrate or circularize. A late defect ofviral DNA import, most probably affecting translocation through the NPC,is demonstrated by fractionation of infected cells and directvisualization (FISH) of intracellular viral DNA. The triplex defectivelinear DNA molecules associate with the nuclear membrane.

The invention focuses on the analysis of cPPT mutant viruses, which arecharacterized by the absence of a central DNA triplex. Most of theexperiments presented herein were also conducted with previouslydescribed CTS mutant virus (Chameau et al., 1994), with the same results(data not shown). In the CTS mutant virus, reverse transcriptionproduces linear DNA molecules containing larger, randomly distributedplus strand overlaps, as compared to the discrete central DNA triplex ofthe wild-type virus. Thus, not only the presence of the DNA triplex, butalso its structural integrity, is important for the nuclear import ofHIV DNA.

The invention shows that the DNA triplex is operative in the context ofan HIV-1 based vector system. Its insertion into a vector devoid of thetriplex reverts a strong defect of nuclear import of the vector DNA towild-type levels of nuclear import.

The central DNA triplex is a common nuclear import determinant oflentiviruses. The location of the central DNA triplex has been preciselydefined in the case of HIV-1. Central strand displacement starts at thefirst nucleotide following the cPPT sequence (Chameau and Clavel, 1991)and stops in general 99 nucleotides downstream, at the ter2 site of theCTS sequence (Chameau et al., 1994). The three dimensional configurationof the three DNA strands of the triplex is as yet unknown. Nevertheless,the presence of a DNA triplex at the center of the genome can begeneralized to all lentiviruses. A central copy of PPT is a commonfeature of all lentiviral genomes and a putative CTS terminator element,revealed by the presence of (A) n and (T) n tracts, also existsapproximately 100 nucleotides downstream (Chameau et al., 1994). Thecentral DNA triplex of the ungulate lentivirus EIAV has beencharacterized recently (Stetor et al., 1999). A central stranddiscontinuity in VISNA virus DNA, referred to as a gap, but mostprobably a nick resulting from the central strand displacement, wasrevealed by S 1 nuclease cleavage (Harris et al., 1981). Sincemitosis-independent replication has been described for most lentiviruses(Gartner et al., 1986; Thormar, 1963), the role of the DNA triplex innuclear import described here for HIV-1 can be generalized to alllentiviruses.

Without being limiting, the invention provides a mechanistic hypothesisfor the role of the central DNA triplex in HIV-1 nuclear import asfollows: A three stranded DNA structure acting as a cis-determinant ofits nuclear import is a novel biological phenomenon with no knowncellular or viral counterparts. Any hypothesis of a molecular mechanismdescribing the role of the central DNA triplex in HIV nuclear import istherefore speculative. The central triplex could act as a viraldeterminant for initiation of the uptake of the HIV DNA filament throughthe nuclear pore. This could be achieved through direct interaction ofthe DNA triplex with components of the pore, or alternatively throughinteraction of the triplex with cellular or viral proteins which shuttlebetween the cytoplasm and the nucleus of the host cell and could dragthe HIV genome into the nucleus. Translocation of the 9.7 kb HIV genomethrough a nuclear pore of maximum diameter of 26 nm must occur in aspecific orientation, after recognition of one extremity of the HIV-1DNA filament to initiate the uptake. A similar situation arises in thenuclear export of messenger RNA, where uptake of the RNA filamentthrough the pore is guided by the 5′Cap structure (Hamm and Mattaj,1990).

Although the conformation of HIV-1 PICs is not well known, it has beenestablished that the extremities of the linear DNA are bridged together,probably after dimerization of the integrase proteins bound at the tipsof the LTRs (Miller et al., 1997). Interestingly, the cPPT and CTScis-active sequences are found at a central position in all lentiviralgenomes. One logical structure for lentiviral PICs would be a double DNAfilament, symmetrically folded on either side of the central triplex bythe integrase dimerization (FIG. 6B). The triplex would then constituteone apex of a filamentous HIV-1 PIC and the integrase dimer the oppositeapex. In cPPT mutant PICs, the absence of a DNA triplex would lead totheir lack of recognition by the nuclear pore machinery or the shuttlingproteins. Identification of the protein ligands of the central DNAtriplex promises to be of primary importance both for our understandingof HIV-1 PIC nuclear import and for the eventual development of drugstargeting this step of HIV replication.

Another mechanistic hypothesis to explain the properties of triplexmutant viruses would involve a defect in the maturation of HIV capsidsinto PICs, prior the translocation of viral DNA into the nucleus.According to this model, triplex defective viral DNA would remaintrapped as integral viral capsids, unable to translocate. Retroviralreverse transcription does not take place at high dilution of the viralcomponents in the cytoplasm of infected cells, but requires thestructural environment of a reverse transcription complex wherecomponents are confined in a capsid protein assembly. The HIV capsidsize exceeds the maximum exclusion diameter of a nuclear pore (Dworetzkyet al., 1998; Gelderblom, 1991). Therefore, before viral DNA can enterthe nucleus, the HIV reverse transcription complexes must undergomaturation into PICs of size compatible with translocation through thenuclear pores (Karageorgos et al., 1993). The maturation of viralcapsids prior to nuclear translocation is well established in severalother viral systems in which the replicative cycle involvestranslocation of the DNA genome through the nuclear membrane of the hostcell (Whittaker and Helenius, 1998). Whereas reverse transcriptionwithin viral capsids has been physically demonstrated for MLV (Bowermanet al., 1989), this has not yet been possible for HIV-1 possibly due tothe fragility of HIV-1 capsids (Borroto-Esoda and Boone, 1991). The HIVreverse transcription complex contains numerous copies of RT polymerase(about 30 to 50 per capsid) (Panet et al., 1975). Owing to the importantdistribution of HIV-1 reverse transcriptase, a high stoichiometry ofenzyme to viral RNA template is necessary to overcome a number oflimiting steps of reverse transcription such as strand transfers orpolymerization pauses during plus and minus strand synthesis andaccurate formation of the central triplex (Klarmann et al., 1993;Chameau et al., 1994). This strongly suggests that central termination,the last event of lentiviral reverse transcription, occurs within anintegral capsid structure. Central termination, which marks the end ofviral DNA synthesis, could be a required signal for viral DNAdecapsidation and its subsequent translocation into the nucleus.

These two putative molecular mechanisms for the DNA triplex mediatednuclear import of HIV-1 are not mutually exclusive. The formation of acentral DNA triplex could trigger the maturation of viral capsids intoPICs, thus making the DNA triplex accessible to shuttling proteins.

The fact that the integrity of the central DNA triplex is required forentry of the HIV-1 genome into the host cell nucleus implies that theentire process of DNA synthesis, including the last central stranddisplacement event, is completed prior to translocation of the HIV PICthrough the nuclear pore. The subcellular distribution of lentiviralreverse transcription is currently under debate and whether HIVreplication occurs in a specific cellular compartment is still an openquestion. On the basis of fractionation studies, it has been reportedthat HIV reverse transcription can occur entirely within the cellnucleus (Bukrinsky et al., 1993a). However, fractionation techniques donot distinguish between an intranuclear localization and associationwith the nuclear membrane. Other authors have proposed on the contrarythat association of the reverse transcription complex with thecytoskeleton is a prerequisite for viral DNA synthesis (Bukrinskaya etal., 1998). Kinetic studies of the synthesis of HIV-1 DNA and itsassociation with the nuclear fraction indicate that the latter processis much more rapid than the former. The synthesis of HIV-1 DNA in thecourse of a single cycle of reverse transcription only reaches a plateau24 to 48 hours following infection, whereas more than 95% of HIV-1 DNAfractionates with the nuclei of infected cells as early as 4 to 6 hoursafter infection (Barbosa et al., 1994). Therefore, we favor a thirdpossibility that lentiviral reverse transcription takes place mainly inthe immediate vicinity of the nuclear membrane and the NPCs, within theviral capsid, although further experiments will be necessary to confirmthis hypothesis.

Cell mitosis does not provide an alternative pathway for the entry oftriplex defective viral DNA into the host cell nucleus. The presentExamples show that central triplex mutant viruses are strongly hamperedin their replication capacity not only in non-dividing but also individing target cells. Conversely, insertion of a DNA triplex sequencein a HIV-1 based vector stimulates gene transduction in both dividingand non-dividing cells. This differs from the published phenotype ofMA/Vpr mutant viruses, where a replication defect has been describedexclusively in non-dividing cells (Bukrinsky et al., 1993b; Heinzingeret al., 1994; von Schwedler et al., 1994). Thus, MA/Vpr HIV mutantswould behave like mitosis-dependent oncoviruses. One possibleexplanation for the different behavior of central triplex mutants isthat these viruses are defective in a late step of the nuclear importprocess, consequently, the triplex deficient DNA molecules associatewith the nuclear membrane. This close association persists duringmitosis. The mutant viral DNA could be trapped within mitotic nuclearmembrane vesicles, where it is unable to reach the cellular chromosomes,such as reported in the case of NLS-LacZ proteins (Bonnerot et al.,1987). Mutations in cellular NLS, inhibiting interactions withkaryopherins, however, are known to induce cytoplasmic accumulation ofthe mutated protein (Kalderon et al., 1984; Lanford and Butel, 1984).Thus, mutations in the NLS sequences contribution to the karyophilicproperties of HIV-1 PICs should induce cytoplasmic retention of theviral DNA, as previously suggested (Gulizia et al., 1994). It istherefore possible that PICs from MA/Vpr mutant viruses reach thecellular chromosomes following disruption of the nuclear membrane duringmitosis. Nevertheless, there is as yet no direct experimental evidencefor a mitosis dependent nuclear import pathway of lentiviral DNAgenomes. As the published phenotype of MA/Vpr mutant viruses must beviewed with some caution, the same caution must be applied to thestarting hypothesis that a nuclear import deficiency in lentivirusesshould lead to a replication defect exclusively in non-dividing cells.Whether lentiviruses can adopt a mitosis dependent nuclear importstrategy, or whether the active nuclear import of lentiviral genomesoccurs in both dividing and non-dividing cells, remains an openquestion.

The present invention provides designs for lentiviral vectors. Since theinfection of non-dividing target cells by lentiviruses relies on theiruse of an active nuclear import pathway, it is important to maintain thelentiviral nuclear import determinants in derived vector constructs.

Classical retroviral vector constructs are replacement vectors in whichthe entire viral coding sequences between the LTRs are deleted andreplaced by the sequences of interest. In the case of lentiviralvectors, this classical strategy leads to the deletion of the centralcis-active sequences cPPT and CTS. The important role of the triplex inHIV nuclear import implies that such replacement vector constructs arenot optimal. Thus, insertion of the HIV DNA triplex into the HR GFPreplacement vector (Naldini et al., 1996) enhanced its gene transductionefficiency by complementing a nuclear import defect of the HR vectorgenome to a rate of DNA import close to that of wild type HIV-1.

It is noteworthy that while HIV vectors lacking a DNA triplex were stillcapable of gene transduction (FIGS. 2C, 2D), the residual replication ofviruses mutated in cPPT is absent or extremely low (FIGS. 6A, 6B). Onepossible explanation is that triplex independent nuclear import couldoccur in the case of a small vector genome (about 4 kb for HR-GFP), butthe presence of a DNA triplex would be required for the import of the9.7 kb native HIV-1 genome. In fact, active, if relatively inefficient,nuclear import of DNA molecules as large as 3 to 4 kb has been reported(Hagstrom et al., 1997).

The cis-active sequences responsible for formation of the DNA triplexare found at the center of all lentiviral genomes. This central positioncould have evolved on account of its structural implications for theconformation of PICs, in so far as symmetrical folding of the left andright arms of the linear DNA molecule around the triplex might benecessary for its efficient uptake through the NPCs (FIG. 7B). If thisis true for the virus, then a central position of the DNA triplex mightalso be required for the efficient nuclear import of vector genomes.

EXAMPLES

The invention will be further clarified by the following examples, whichare intended to be purely exemplary of the invention.

Example 1 Experimental Procedures. Cells

MT4 cells are HTLV-1 transformed human CD4+ T cells that allow acutecytopathic HIV-1 infection (Harada et al., 1985). H9 cells are lesspermissive to HIV but allow chronic production after infection. MT4 andH9 cells were maintained in RPMI 1640 medium supplemented with 10% fetalcalf serum (FCS). Peripheral blood lymphocytes (PBLs) were obtained fromhealthy donors, stimulated with I pg/ml of phytohemagglutinin(Wellcome), and maintained in the presence of Interleukin-2 (10%lymphocult ; Biotest Diagnostics). 293T cells were grown in DMEM mediumsupplemented with 10% FCS. P4 indicator cells are HIV infectible HeLaCD4+ cells carrying the LacZ gene under the control of the HIV-1 LTR(Chameau et al., 1994). P4 cells are grown in DMEM medium supplementedwith 10% FCS and 500 μg/ml of G418.

Collection and Fractionation of Cells.

Cord Blood samples were collected with the informed consent of themothers. CD34+ cells were purified as previously described (Robin, C. etal., 1999) using miniMACS immunomagnetic bead separation system (MiltenyBiotec). The purity of bead-separated CD34+ cells was over 75%.CD34+CD381o/-fractions were further purified by cell sorting with a FACSVantage™ equipped with an argon ion laser (Becton Dickinson), usingmurine monoclonal antibodies (MoAbs) directed against CD34-PE-Cy5(Immunotech) and CD38-PE (Becton Dickinson). CD34+ cells were eitherfrozen in fetal calf serum (FCS, Stem Cell) containing 10% DMSO (Sigma)or used immediately.

DNA Constructs

Proviral Plasmids:

Site directed mutagenesis was performed as previously described (Kunkel,1985) in M13mp18 carrying anEcoRI 1.1 kb insert (4684 to 5779) from theinfectious molecular clone pLAI3. Mutagenic primers were as follows:

cPPT-AG (SEQ ID NO: 1) 5′pCAATTTTAAAAGAAGAGGGGGGATT 3′ cPPT-D:(SEQ ID NO: 2) 5′pATTCATCCACAACTTCAAGCGCCGCGGTGGTATTGGGGGGTAC 3′.

pcPPT-AG, pcPPT-D, pcPPT-25 and pCTS were constructed by cloning backthe mutated EcoRI fragment into pLAI3.

Vector Plasmids:

Vector plasmids were derived from HR′CMVLacZ (Naldini et al., 1996). TheLacZ reporter gene was replaced by the EGFP gene (Clontech). EGFP genewas amplified by PCR using Pfu polymerase (Stratagene) from pEGFP-N 1plasmid, adding BamHI and Xhol restriction sites at the 5′and 3′endsrespectively. PCR primers were as follows:

(SEQ ID NO: 3) Bam GFP: 5′CC GGATCC CCA CCG GTC GCC ACC 3′(SEQ ID NO: 4) Xho GFP: 5′CC CTCGAG CTA GAG TCG CGG CCG 3′.

The HR GFP vector was constructed by cloning back this PCR fragment intothe BamHI and XhoI sites of pHR'CMVLacZ, replacing the LacZ ORF withEGFP. TRIP ΔU3 CMV GFP and TRIP ΔU3 PL CMV GFP:

First, a subclone containing a unique LTR was constructed and named pUCLTR. The Kpnl IXbal fragment of TRIP GFP encompassing its 3′LTR wascloned into pUC 18. Then the EcoRI site was destroyed by filling in,creating the vector pUC LTR RI-. Diverging PCR was performed on pUC LTRRI-with the aim of amplifying the whole plasmid except the promotor andthe enhancer of the U3 sequence. The primers were:

DU3−: (SEQ ID NO: 5) 5′CGGAATTCGGATCCGCGGCCGCATCGATCTTGTCTTCGTTGGGAGTG3′ DU3+: (SEQ ID NO: 6) 5′CGGAATTCAGCCGTCTCGAGAGATGCTGCATATAAGCAGC 3′.

The primers contain the restriction sites to be inserted instead of theU3 sequence including the EcoRI site present on each primer. The PCRproduct was digested with EcoRI then used to transform competentbacteria. The plasmid constructed thereby was named pLTR EU3 RI-.

The polylinker inserted instead of theU3 sequence in pLTR ΔU3 RI-is:Clal-Notl-BamHI-EcoRI-Mlul Xhol.

The TRIPΔU3 PL GFP plasmid was constructed by replacing the Kpnl/Nhelfragment of TRIP GFP containing the 3′LTR with the KpnllXbal fragment ofTRIP GFP containing the 3′LTR with the Kpnl/Xbal fragment of pLTR ΔU3RI-(Nhel and Xbal restriction products are compatible) Then thepolylinker (PL) was deleted from TRIPΔU3 RI- by digestion with Clal/Xholand filing in.

The TRIP ΔU3 GFP plasmid was constructed by exchanging the Kpnl/Nhelfragment of TRIP GFP with the Kpnl/Xbal fragment of pLTR U3 ΔPL RI-.

A 178 bp fragment of pLAI3 (4793 to 4971), encompassing cPPT and CTS,was amplified by PCR. Nan restriction sites were added in 5′ of theprimers with the aim of inserting this fragment into the unique Clalsite of HR GFP:

Nar TRIP+: (SEQ ID NO: 7) 5′GTG GTC GGCGCC GAATTC ACA AAT GGC AGT ATT CAT CC 3′ Nar TRIP−:(SEQ ID NO: 8) 5′ GTC GTC GGCGCC CCA AAG TGG ATC TCT GCT GTC C 3′

Insertion of this triplex sequence in the correct orientation gave riseto the TRIP GFP plasmid vector, and TRIPinv GFP in the reverseorientation. Alternatively, the same triplex fragment was amplified frompcPPT-AG, pcPPT-D, pcPPT-225, and pCTS plasmids to generate vectorsincluding the same mutations in the cPPT or in the CTS as thecorresponding viruses.

TRIP EF1αGFP and TRIPΔU3 EF1α GFP:

The CMV promotor of TRIP GFP was replaced by the EF1Iα promotor. Thetriplex sequence and the EFIα promotor were first amplified separately,with overlapping primers. The triplex sequence was amplified with theprimers Nar TRIP+ and Mlu TRIP-on the matrix pLai and the EFIα promotorwas amplified on the matrix pEFpgkneo with the primers Mlu EF1+ and BamEF1−.

Nar TRIP+: (SEQ ID NO: 9)5′GTC GTC GGCGCC GAATTC ACA AAT GGC AGT ATT CAT CC 3′ MluTRIP−:(SEQ ID NO: 10) 5′AGC CTC ACG ACGCGT AT CAG CCA AAG TGG ATC TCT GCT G 3′Mlu EF1+: (SEQ ID NO: 11) 5′CTG AT ACGCGT CGT GAG GCT CCG GTG 3′Bam EF1−: (SEQ ID NO: 12) 5′CG GGATCC TGT GTT CTG GCG GCA AAC3′

Then a second round of PCR was performed on a mixture of the first twoPCR products, using the external primers Nar TRIP+ and Bam EF1−. Thetriplex sequence and the EF1α promotor stuck together by this technique.

Plasmid TRIP EF1α GFP was constructed by replacing the EcoRI/BamHIfragment of TRIP GFP containing the triplex sequence and the CMVpromotor by the PCR product TRIP EF 1α digested with EcoRI/BamHI (theNar TRIP+ primer possesses a EcoRI site in its 5′end).

To construct the TRIP ΔU3 EFIα GFP plasmid, the EcoR/BamHI fragment ofTRIP EF1αGFP containing the triplex sequence and the EFIα promotor wasinserted in TRIP ΔU3 GFP instead of the EcoRI/BamHI fragment containingthe triplex sequence and the CMV promotor.

Virus and Vector Production

For the investigations reported in FIGS. 1-6, viruses were produced bytransient transfection of HeLa cells by the calcium phosphateco-precipitation technique. Vector particles were produced by transientco-transfection of 293T by the vector plasmid, and encapsidation plasmid(p8.2) and a VSV envelope expression plasmid (pHCMV-G, (Yee et al.,1994)), as previously described (Naldini et al., 1996). All virus andvector supernatants were treated with DNasel (1 μg/ml in the presence of1 μM MgCl₂) for 15 minutes at 37° C.

Lentiviral Vector Particle Production

For the investigations reported in FIGS. 7-9, viruses were produced bytransient co-transfection of 293T by the vector plasmid, anencapsidation plasmid (p8.2) and a VSV envelope expression plasmid(pHCMV-G, (Yee et al., 1994)), as previously described (Naldini et al.,1996). All virus and vector supernatants were treated with DNaseI (1μg/ml in the presence of 1 μM MgCl₂) for 15 minutes at 37° C.

Virus and Vector Titrations

For the investigations reported in FIGS. 1-6, one cycle titration ofviruses were performed in triplicate by infection of P4 cells plated in96 well plates, with equivalent amounts of particles (1 ng of p24 viralantigen per well), in the presence of 20 μM of DEAE-dextran. Theprotease inhibitor Saquinavir (Roche), was added (1 μM) throughout theexperiment, to restrict the analysis to a single cycle of infection.Cell mitosis was inhibited by aphicolin treatment (8 uM), the day priorto infection. The β-Galactosidase activity was measured 48 hours afterinfection using a chemiluminescent β-Gal reporter gene assay(Boehringer).

HeLa cells were infected in triplicate with equivalent amounts of vectorparticles (5 ng P24 per well). At 48 hours post transduction, the mediumwas replaced by 200 μl of TNB (Tris 50 mM pH 7.5, NaCl 150 mM) andfluorescence of living cells was quantitated using a microplatefluorimeter (Victor², Wallac) and EGFP adapted filters (excitation: 485nm, emission: 520 nm).

Transduction Protocol

For the investigations reported in FIG. 7-9, 24- or 96-well tissueculture plates were coated with fibronectin (Bio-Whittaker Europe)according to the manufacturers instructions. Human CD34+ populationswere plated, immediately after purification or thawing, at 2 to 3×10⁵cells/ml in serum free medium (IMDM containing 11.5 μM a-thioglycerol,1.5% BSA (both from Sigma), sonicated lipids and iron-saturated humantransferrin) or a-MEM containing 10% FCS in presence of 4 μg/ml ofpolybrene (Sigma) and 4 recombinant (r) human (hu): rhu-Stem Cell Factor(SCF, 100 ng/ml, provided by Amgen), Flt3-Ligand (FL, 100 ng/ml,Diaclone), IL-3 (60 ng/ml, Sandoz), and pegylated-(PEG-)rhu-Megacaryocyte Growth and Differentiation Factor (MGDF) (10 ng/ml,Amgen), and concentrated lentiviral virus at the concentration of 100 ngof viral P24/ml during 24 hours. Cells were then washed and cultured inlympho-myeloid conditions (in culture tissue culture plates precoatedwith MS5 cells in RPMI supplemented with 10% human serum, 5% FCS and thefollowing 7 cytokines: rhu-SCF (50 ng/ml), rhu-FL (50 ng/ml),PEG-rhu-MGDF (50 ng/ml), rhu-IL-3 (10 ng/ml), rhu-IL-2 (5 ng/ml),rhu-IL-15 (10 ng/ml), and rhu IL-7 (20 ng/ml) (the three IL-being fromDiaclone) for 48 hours. Then, expression of eGFP in the CD34+ cellfraction was evaluated using a CD34-PE-Cy5 MoAb (Immunotech). Analysiswas done on a FACS Scan using the Cellquest software (Becton Dickinson).

Clonogenic and Long Term Culture (LTC) Assays

Clonogenic progenitors from human fresh CB cells were assayed in 0.8%methylcellulose containing 30% FCS, 1% deionized BSA, and 10-4M2-mercaptoethanol, in the presence of 50 ng/mlrhu-SCF, 10 ng/ml rhu-GCSF(Amgen), 2 ng/ml rhu-IL3, and 2U/ml rhu-EPO (Amersham). Bone marrow (BM)cells from engrafted NOD-SCID mice were plated in the presence ofrhu-SCF,-IL-3,-EPO, and-GM-CSF (10 ng/ml) as described (Pflumio, F. etal., 1996). Progenitors were scored on day 14-16 according to criteriaalready described (Croisille L. et al., 1994) and EGFP expressionobserved by fluorescent microscopy using a Nikon Eclipse TE300microscope. Long Term Culture (LTC) was performed as previouslydescribed (Issaad C. et al., 1993) either in limiting dilution in96-wells plates using the FACS vantage equipped with an ACDU (BD) or inbulk culture in 24-wells plates containing a confluent layer of themurine stromal cell line MS5. After 5 to 10 weeks, adherent andnon-adherent cells were harvested and plated for the clonogenic assay.For both clonogenic and LTC-IC assays, colonies were picked individuallyand frozen before PCR analysis.

PCR Analysis

PCR analysis was performed on genomic DNA obtained either from coloniesderived from clonogenic progenitors or from clones grown inlympho-myeloid cultures. Cells were lysed and proteins were digested in20p1 of buffer containing proteinase K (10 μg/ml), KCl (50 mM), Tris-HCl(10 mM, pH 8.3), MgCl₂ (2.5 mM), gelatin (0.1 mg/ml), NP40 (0.45%), andTween 20 (0.45%). Amplification of genomic DNA was performed with thesense primer 5′CCCTCGAGCTAGAGTCGCGGCCG 3′ (SEQ ID NO: 13) and theantisense primer 5′CCGGATCCCCACCGGTCGCCACC 3′ (SEQ ID NO: 14) at theannealing temperature of 62° C. The amplification resulted in an 800 bpproduct.

Viral and Vector DNA Analysis

P4 or MT4 cells were infected at a high multiplicity of viruses (150 ngof p24 per 106 cells) or transduced by vectors (25 ng of p24 per 10⁶cells), in the presence of 20 μg/ml of DEAEdextran in the case of P4cells. DNA from infected or transduced cells was extracted at varioustimes, restricted, and analyzed by Southern blot. In all cases,contaminating bacterial plasmid DNA was removed from the analysis byDpnl digestion. DNA from infected cells was digested by Mscl and Xholand DNA from transduced cells by EcoNI, AvaJI and Xhol. Afterelectrophoresis and transfer of 10 μg of digested DNA, membranes werehybridized with random primed [³²P]-labeled DNA probes (Rediprime II,Amersham). Virus specific DNA probe was amplified by PCR from pLAI3plasmid template using the following primers:

(SEQ ID NO: 15) 5Msc: 5′AGA AGA AAT GAT GAO AGC ATG 3′ (SEQ ID NO: 16)3Msc: 5′TGC CAG TTC TAG CTC TG 3′.

The resulting 1680 bp DNA fragment (from position 1818 to 3498 of pLAI3)overlaps the Mscl restriction site at position 2655 of viral genomes.

Vector probe was synthesized by PCR on pTRIP GFP with the primers:

(SEQ ID NO: 17) 5EcoNI: 5′CAG GGA CTT GAA AGC GAA AG 3′ (SEQ ID NO: 18)3EcoNI: 5′GCT TGT GTA ATT GTT AAT TTC TCT GTC 3′

The vector probe is a 1027 bp fragment (from position 649 to 1676 ofpTRIP GFP) and overlaps the EcoNI site at position 1156 of vectorgenomes.

To assay the amount of retrotranscribed DNA from wild type and PPT-AGand cPPT-D viruses, a similar protocol was followed except that the DNAextracted at 12 hours post-infection was restricted by Mscl and Dpnl.The probe used for hybridization was the Mscl 1.9 kb internal fragmentfrom pLAI3. Hybridization signals were quantitated using aphosphorimager (Molecular Dynamics) and the ImageQuant software.

In Situ Hybridization

P4 cells were infected at a high multiplicity (2 μg of p24 antigen ofeach virus per 10⁶ cells), in the presence of 20 μg/ml DEAE dextran. At24 hours post-infection, cells were trypsinized, extensively washed (inorder to remove viral particles adsorbed in the plasma membrane), andre-plated on glass cover slides in 24 well plates. Cells were grown fora further 48 hours and fixed in 4% PFA/PBS for 20 minutes at roomtemperature. Cells were washed in PBS and permeabilized by 0.5%Triton/0.5% Saponin in PBS, for 5 minutes at room temperature.Dehydrated samples were treated with RNase A (200 μg/ml in 2×SSC), onehour at 37° C. and by proteinase K (6 μg/ml in PBS), about 5 minutes.Samples were denatured by incubation in 70% deionized formamide/2×SSCfor 2 minutes at 70° C. followed by 30% deionized formamide/2×SSC for 2minutes at 70° C. Hybridizations were performed overnight at 37° C.using a nick translated biotynilated pLAI3 plasmid (50% deionizedformamide, 10% dextran sulfate, 10 μg/mi Salmon sperm DNA, 0.1% Tween 20in 2×SSC). Samples were extensively washed (serial washing in 2×SSC/50%formamide at room temperature and then at 50° C.). Detection ofhybridized probes was performed using the Tyramid-StreptavidinTSA-Direct kit (NEN) according to the manufacturer's instructions.

Example 2 Central Initiation of Reverse Transcription is an EssentialStep of the HIV-1 Replicative Cycle

In a previous work, we showed that conservative mutations in the cPPTand CTS sequences severely impaired virus replication (Charneau et al.,1992; Hungnes et al., 1992). A central initiation mutant virus(cPPT-225) and a termination mutant virus (CTS) showed respectively fourfold and ten fold decreased infectivity in one round titrationexperiments. In order to inactivate the function of the cPPT,semi-conservative mutations were introduced in the overlapping integrasecoding region. In the mutant virus cPPT-D, the lysine to arginine changeat position 188 allowed the introduction of a total of 10 mutations intothe 19 nucleotide sequence of the PPT primer (FIG. 1A) (Huber andRichardson, 1990). The effect of this amino acid change on virusreplication was checked by construction of the control cPPT-AG mutantvirus, in which a single mutation from purine to purine, respecting thepolypurine nature of cPPT, induced the same amino acid change. Thepresence of a DNA triplex in retrotranscribed wild type and cPPT-AGviruses DNA, and its absence from cPPT-D virus DNA, was confirmed by S 1nuclease cleavage of Hirt DNA from infected cells as previouslydescribed (Harris et al., 1981; Chameau and Clavel, 1991).

Virus infectivity was first evaluated in classical kinetic replicationexperiments in cell cultures. PHA stimulated peripheral bloodlymphocytes (PBLs) and MT4 cells were infected with equal numbers ofviral particles, normalized according to the capsid protein (p24)content of the viral supernatants, and reverse transcriptase activitywas followed over time in the culture supernatants (FIG. 1 B). Growthcurves of the wild type HIV-1 LAI and cPPT-AG control viruses weresimilar in both cell systems. The fact that the K188R mutation occursnaturally in some HIV-1 isolates, already suggested that it has littleor no effect on the integrase and PPT functions. In contrast, when PBLswere infected with the cPPT-D mutant virus, no replication was detectedduring the 15 days of culture. The same was true for MT4 cells, despitetheir high susceptibility to HIV infection. The cPPT-D mutant virus wasalso non infectious in immortalized cell lines such as H9 or CEM (datanot shown).

Virus infectivity was then quantitatively analyzed by titrations basedon a single round of replication (FIG. 1C). P4 indicator cells (HeLa CD4LTR-LacZ) (Chameau et al., 1994) were infected with equivalent numbersof virus particles of the different viruses. These one cycle titrationsconfirmed the almost complete loss of infectivity of the cPPT-D mutantvirus. In P4 cells, infectivity of the cPPT-AG control was identical tothat of the wild type virus, whereas infectivity of the cPPT-D mutantwas strongly reduced, to levels close to background. The same resultswere obtained in aphidicolin treated, non dividing P4 cells (Figure IC,right panel).

These findings strongly suggest that the central initiation of reversetranscription is necessary for HIV replication in non dividing as wellas in proliferating cells.

Example 3 Virus Production is Not Affected by Mutations in cPPT

We checked that the different mutations introduced into the cPPT-AG andcPPT-D plasmid proviruses did not affect the late steps of thereplicative cycle. Virus production was quantified, according to the P24content of the supernatants, after transient transfections of HeLa cellsby proviral plasmids. The production of the cPPT mutant viruses wasfound not to be significantly different from that of the wild type virus(FIG. 2A). Hence the mutation K188R does not affect the late phase ofHIV-1 replication. Therefore, the defective step involved in thephenotype of the cPPT-D mutant virus must precede the expression ofviral DNA and belong to the early phase of the HIV replicative cycle.

Example 4 Mutations in the cPPT Do Not Affect the Rate of ReverseTranscription of HIV-1 Genome

The effect of mutations in cPPT on viral DNA synthesis was evaluated byquantifying the DNA synthesized in a single round of retrotranscription(FIG. 2B). An internal Mscl restriction fragment from the viral DNA ofinfected cells was detected by Southern blotting and quantitated, usingthe corresponding Mscl DNA fragment as a probe. Since the internal Msclfragment is common to the integrated proviral DNA and the unintegratedlinear and circular molecules, its quantitation reflects the totalamount of viral DNA, irrespective of its integrated or unintegratedstate. To limit the analysis to the first cycle of reversetranscription, DNA from infected P4 cells was harvested 12 hours afterinfection, before initiation of a second round of infection. The totalamount of DNA retrotranscribed in a single cycle of reversetranscription was the same after infection with the cPPT-D mutant, thecPPT-AG control or the wild-type virus. These experiments showed that,whereas mutations in cPPT abolish virus replication, they do not affectthe rate of DNA synthesis. The replicative defect of cPPT mutant virusesimplicates a step subsequent to viral DNA synthesis.

Example 5 Lack of a Central DNA Triplex Does Not Affect the In VitroIntegration of HIV-1 PICs

The in vitro integration ability of PICs from wild-type HIV-1 andcentral initiation mutants was compared (FIG. 2C) using a quantitativein vitro integration assay as described by Farnet (Farnet and Haseltine,1990), with minor modifications. Since HIV-1 replication complexesreside only transiently in the cytoplasm of freshly infected cells(Barbosa et al., 1994), the preparation of sufficient amounts of HIVPICs requires massive infection and cellular fractionation within 4 to 6hours. This was achieved by co-culture of H9 cells chronically infectedby either wild type or cPPT-225 virus and uninfected HUT 78 targetcells. The cPPT0225 mutant virus (FIG. 1A) was chosen for theseexperiments instead of the non infectious cPPT-D, which is unable toestablish a chronic infection. The residual infectivity of cPPT-225mutant virus is low but sufficient to allow it to slowly propagate incell cultures (Chameau et al., 1992).

HIV PICs were isolated from the cytoplasm of infected cells andincubated in the presence of a linearized Bluescript plasmid target DNA.Integration was revealed by the presence of a 12.7 kb fragment, reactiveto the HIV-1 probe, corresponding to the expected size of the 9.7 kblinear HIV genome integrated into the 3 kb target DNA.

The amount of linear DNA integrated into the plasmid DNA did not differbetween the wild type and cPPT 225 mutant virus (FIG. 2C). Hence HIV-1PICs from the cPPT mutant retained their full ability to integrate invitro. The defective replication step of central DNA triplex mutantviruses must lie after reverse transcription but before integration oftheir linear HIV genome into the host cell chromatin.

Example 6 Impaired Nuclear Import of Central DNA Triplex Mutant Viruses

The foregoing experiments suggested that the replicative defect ofcentral DNA triplex mutant viruses was related to the access of HIV PICsto chromatin of the cellular target. Hence we tested the hypothesis of anuclear import defect of DNA from cPPT mutant viruses. Studies on thenuclear import of HIV-1 PICs are hampered by lack of a quantitative andreproducible assay for nuclear import at the level of the viral DNA.Once retrotranscribed in the cytoplasm, the retroviral linear DNA isimported into the nucleus where it either integrates or circularizes.Unintegrated retroviral DNA circles, containing one or two LTRs, arefound exclusively within the nucleus, and thus represent convenientmarkers of viral DNA nuclear import. To assess HIV DNA nuclear import,previous studies used PCR amplification of two unintegrated LTR DNAcircles. However, as reported herein and by Barbosa et al., (1994),because two LTR circles represent a minute fraction of the HIV DNA ininfected cells, their detection is very sensitive to minor alterationsof cell physiology or virus infectivity. Therefore, we designed a novelassay which permits a quantitative follow-up by Southern blot of thesynthesis, circularization, and integration of HIV DNA.

Briefly, DNA from infected cells is prepared at various time points anddigested with Mscl, a restriction enzyme which cuts the HIV-1 genometwice. Using a PCR generated DNA probe exactly overlapping the 5′Msclsite, several specific bands are revealed (FIG. 3A). The internal 1.9 kbMscl fragment is common to all viral DNA species irrespective of theirintegrated or unintegrated state and quantitation of this band indicatesthe total amount of viral DNA in infected cells. A 2.6 kb bandcorresponds to the distal 5′Mscl fragment of unintegrated linearHIV-DNA. To minimize transfer bias due to the large size of DNA circlesspecific fragments, the DNA is further cut with Xhol. One and two LTRcircular DNA then appears at 2.8 and 3.4 kb bands respectively. Sincethe DNA probe exactly overlaps the 5′Mscl site, the intensity of eachband is directly proportional to the quantity of the corresponding viralDNA species. The amount of integrated proviral DNA is calculated bysubtracting from the total amount of viral DNA the signals ofunintegrated linear and circular viral DNAs. A parallel quantitation ofthe same infected cell population was performed after a Hirtfractionation to separate low molecular weight unintegrated viral DNAfrom high molecular weight integrated proviral DNA. This gave rise toidentical results, thus validating the one step subtractive calculation.

As indicated by the kinetics of accumulation of total viral DNA (1. 9 kbinternal fragment), the synthesis of viral DNA proceeded for 24 to 48hours after infection, reflecting an asynchronous infection process. Theamounts of total viral DNA from cPPT-AG and cPPT-D mutant viruses weresimilar to those of wild-type HIV-1. As previously, the mutations incPPT did not influence the rate of DNA synthesis. Detectable amounts offull length unintegrated linear DNA were present in cells as early as 6hours after infection (FIG. 3B). Integrated proviruses and DNA circleswere first detected 12 hours after infection. Integration andcircularization proceeded to completion over a further 36 hours.

On completion of one cycle of infection, in the case of the wild typevirus, about 55% of the viral DNA had integrated into the host cell DNA,about 35% had circularized into one LTR circle, and a small fraction ofless than 10% remained in the form of stable unintegrated linear DNA(FIG. 3C). Notably, two LTR circular DNA, although detectable at 48hours after infection, was present only in trace amounts. DNA from thecPPT-AG control virus was processed in a very similar manner to DNA fromthe wild type virus.

In the case of the cPPT-D mutant virus, a marked alteration in thepattern of intracellular viral DNA was evident, with a clear andpersistent accumulation of unintegrated linear molecules. At 48 hoursafter infection, only very small amounts of one LTR circular DNA andintegrated proviral DNA had been generated and more than 90% of thecPPT-D mutant DNA remained blocked in the unintegrated linear form (FIG.3C).

A nuclear import defect is expected to decrease the proportion ofnuclear viral DNA species (integrated proviruses and one and two LTRcircles) and to concomitantly increase the proportion of untranslocatedlinear DNA molecules. Thus, the intracellular DNA profile of cPPT-Dmutant virus strongly suggests a defect of viral DNA nuclear import.

Example 7 Linear DNA from Central DNA Triplex Mutant Viruses Accumulatesat the Vicinity of the Nuclear Membrane

To further characterize the nuclear import defect of central DNA triplexmutant viruses, we addressed the questions of whether the mutated linearDNA molecules accumulate in a particular subcellular compartment. Thenuclear import process can be divided into two main phases, docking ofthe nuclear component to the nuclear membrane and its translocationthrough the nuclear pore complex (NPC). We first conducted classicalnuclei/cytoplasm fractionation of infected cells, followed by southernblot detection of viral DNA. The totality of viral DNA of all viruseswas associated with the nuclei of infected P4 cells, 24 hours afterinfection (FIG. 4A), suggesting that docking of HIV DNA to the nuclearmembrane was not affected by the lack of a central DNA triplex.

To confirm that central triplex deficient DNA molecules do accumulate atthe nuclear membrane, we used fluorescent in situ hybridization (FISH)to directly visualize the intracellular location of HIV genomes. P4cells were infected at a high multiplicity in one cycle conditions,hybridized with a full length HIV-1 genome probe, and observed bydeconvolution microscopy. Specific dots were found predominantly withinthe nucleus in the case of the wild type and cPPT AG control viruses(FIG. 4B). Since FISH cannot distinguish between the different HIV DNAspecies, these intranuclear viral DNA molecules could have beenintegrated proviruses or unintegrated DNA circles. Some rare genomeswere associated with the nuclear membrane and probably represented theresidual linear DNA detected by Southern blotting at the same time afterinfection. Other dots associated with the plasma membrane most likelyderived from membrane adsorbed defective particles containing partiallyretrotranscribed genomes (Lori et al., 1992). In contrast, HIV genomeswere predominantly localized at the nuclear membrane and almostcompletely absent from the nucleus in the case of the cPPT-D mutantvirus. As the Southern blot DNA profile indicated that practically allcPPT-D DNA was blocked in the linear form, we can assume that these HIVgenomes associated with the nuclear membrane were unintegrated linearDNA molecules. This direct visualization of viral DNA molecules ininfected cells confirmed the association of the viral DNA of centraltriplex mutants with the nuclear membrane.

Our FISH experiments suggest that cPPT mutant viruses are defective inthe translocation of their genome through the NPCs. Nevertheless, theclear demonstration of a translocation defect, by localization of mutantviral DNA at the cytoplasmic side of NPCs, is not accessible throughFISH experiments.

Altogether, we conclude from these results that the central DNA triplexof HIV-1, created by central initiation and termination steps duringreverse transcription, is important for HIV-1 PICs to enter the hostcell nucleus. In the absence of DNA triplex, viral DNA nuclear import isseverely impaired at a stage immediately preceding or during thetranslocation of HIV-1 DNA through the nuclear pore.

Example 8 Impact of the Central DNA Triplex on Gene Transduction by anHIV-1 Based Vector System

Having identified the central DNA triplex as a key determinant for thenuclear import of HIV-1, we tested the effect of inserting the centralcis-active sequences of HIV-1 into the previously described HR HIV-1vector (Naldini et al., 1996) (FIG. 5A). To monitor gene transduction, agene encoding the green fluorescent protein (GFP) was further inserted.The vector containing a DNA triplex sequence was called TRIP GFP.Controls included similar constructs with mutated cPPT or CTS and a wildtype central region inserted in reverse, non-functional orientation(TRIPinv GFP). The presence of a DNA triplex in retrotranscribed TRIPGFP vector DNA, and its absence from HR GFP, TRIPinv GFP, and fromvectors containing a mutated version of the central triplex sequence,was confirmed by 51 nuclease cleavage of Hirt DNA isolated fromtransduced cells.

The number of vector particles produced by transient transfection wasnormalized prior to transduction according to the levels of capsidprotein (p24), reverse transcriptase activity, and the quantity ofgenomic vector RNA in transfected cell supernatants. Similar productionof the various vectors was obtained, with a linear correlation betweenthe three normalization criteria (data not shown). Hence insertion ofthe central region of the HIV-1 genome into the HR vector did notinfluence the rate of genomic RNA encapsidation. Dividing ornon-dividing (aphidicolin treated) HeLa cells, were transduced withequivalent numbers of HR-GFP or TRIP-GFP vector particles and GFPexpression was monitored 48 hours later by fluorescence quantitation.Pseudotransduction of GFP activity due to the direct delivery of GFPproteins to target cells by the fusing vector particles was calculatedfrom the transduction of cells treated with an HIV-1 RT inhibitor (1 μMNevirapin, Boehringer Ingelheim) and this background subtracted from thefluorescence signal. Non-specific fluorescence due to secondarytransfection caused by calcium/phosphate DNA co-precipitate in thevector supernatants was eliminated by treating the vector stock withDNasel prior to transduction.

Under these conditions, the presence of the triplex sequence in the HIVvector increased GFP transduction in HeLa cells by more than ten fold(FIG. 5B). A similar enhancement of gene transduction was observed inother target cell lines such as MT4 or 293T (data not shown). Thiseffect was lost if the triplex sequence was inserted in the reverseorientation (Figure SB) or mutated in the cPPT (not shown).

Example 9 The Presence of a DNA Triplex in HIV-1 Vectors Increases theRate of Vector Genome Nuclear Import to Wild Type Levels

It was then of interest to determine whether the increase in GFPfluorescence induced by insertion of a triplex sequence in the HIVvector was due to its effect on the nuclear import of vector DNA. Toaddress this question, we adapted our quantitative Southern blot assayfor intracellular viral DNA to the vector system. DNA from vectortransduced cells was digested with EcoNI and Aval to produce an internal0.8 kb fragment, and with Xhol. Using a PCR generated DNA probe exactlyoverlapping the EcoNI site, signals specific for the unintegrated linearvector genome, and for one and two LTR DNA circles were expected at 1.2kb, 1.4 kb, and 2 kb respectively. The processing of vector DNA wasanalyzed at various time points after transduction of HeLa cells.

The total quantity of vector DNA synthesized in transduced cells wascomparable for vectors containing or lacking the DNA triplex (FIG. 5C).Once again, insertion of the cPPT and CTS sequences, in eitherorientation, into the HR vector did not influence the rate of reversetranscription of its genome. After phosphorimage quantitation, we foundthe intracellular fate of DNA from the HR-GFP and TRIPinv-GFP vectors(Figure SD) to closely resemble that of DNA from central triplexdefective viruses and the fate of DNA from the TRIP-GFP vector to followthat of DNA from the wild type HIV-1 LAI virus (FIG. 3C).

A defect of DNA nuclear import was evident in the case of the HR-GFP andTRIPinv GFP vectors. The intracellular fate of DNA from these vectorswas characterized by a strong accumulation of unintegrated linearmolecules, together with small amounts of integrated provirus and oneand two LTR circles. This DNA profile was strongly reminiscent of thatof the cPPT-D mutant virus. On completion of the processing of vectorDNA in transduced cells, 70 to 80% of the DNA from HR-GFP andTRIPinv-GFP constructs remained in the form of unintegrated linearmolecules, while only 10 to 15% were present as unintegrated on LTRcircles and 5 to 10% as integrated proviruses. This low but detectableamount of integrated vector DNA would account for the gene transductionobtained using HR-GFP or TRIPinv-GFP vectors.

This quantitative assay also showed that insertion of the triplexsequence of HIV DNA into the HR vector in the correct orientationcomplemented its nuclear import deficiency to wildtype levels. The finalstate of TRIP-GFP DNA in transduced cells was similar to that observedwith wild type HIV-1 virus: 50% or more of the vector DNA integrated thechromatin of the target cell, an important fraction circularized and afew molecules remained as unintegrated linear DNA (compare FIGS. 5D and3C). By contrast, insertion of the triplex sequence into the HR vectorupstream of the internal CMV promoter did not influence GFP expressionat the transcriptional level. This was checked by transfection of HeLacells with pHR-GFP, pTRIP-GFP, and pTRIPinv-GFP plasmids andfluorescence quantitation (data not shown).

It may be inferred from these results that the increase in GFPtransduction obtained with the TRIP-GFP vector is entirely imputable tostrong stimulation of its nuclear import by the presence of the triplex.This finding again emphasizes the important role of the HIV DNA triplexin the nuclear import of viral and vector DNA.

Example 10 HIV-1 Vectors Containing the DNA Triplex Sequence Allow anEfficient Gene Transfer in Hematopoietic Stem Cells

FIGS. 7A and 7B illustrate the results of two successful transductionexperiments using CD34+ human cord blood cells. They show that after ashort 24 or 60 hour transduction protocol, respectively 71.5% and morethan 90% of the CD34+ cells strongly express the GFP reporter protein.This expression reflects stable transduction of the cells since viralintegration was confirmed, at least in the same proportions, by PCRassays on DNA extracted from progenitor-derived cell colonies harvested14 days after having seeded cells in clonogenic progenitor assay.Identical results have been obtained using freshly purified CD34+ cellsor the same CD34+ cells that have been frozen after purification andthawed several weeks later for the transduction experiment. We also hadcomparable transduction efficiencies using another hematopoietic stemcell source, peripheral blood mobilized stem cells (PBMC), collected bycytapheresis after cytokine stimulation. CD34+ PBMC were also transducedeither immediately after purification or after a freezing/thawing stepwith identical results. Using long term culture (LTC) and NOD-SCIDrepopulating assays we have shown that we have transduced cells havingmultiple lympho-myeloid potentialities and the ability to repopulateNOD-SCID mouse bone marrow 4 months after graft. These functional assaysrepresent the ultimate experiments available, at the moment, to assesshuman hematopoietic stem cell function.

Example 11 The Presence of the DNA Triplex Sequence in Lentiviral VectorConstructs Strongly Stimulates Gene Transfer Into Hematopoietic StemCells

The dose/response experiment reported in FIG. 8 (representative of 3experiments) was designed to compare transduction efficiency in humanhematopoietic stem cells of HIV-1 vectors including or lacking the DNAtriplex sequence. We first plotted (A) the percentage of CD34+eGFP+cells obtained as a function of the vector concentration used fortransduction. We observe that whatever was the dose of vector, the TRIP+vector was more efficient than the TRIP one, with respectively a mean of4019% and 15.412.5% of CD34+ cells being eGFP+ for 500 ng viral P24/mlof each vector (n=3 exp). A 4-6 fold increase in the final percentage ofGFP+ cells was obtained after transduction by the TRIP-GFP vector whencompared to results obtained after transduction with HR-GFP vector. Thedifference in efficiency between the two vectors is also highlightedwhen the mean of fluorescence intensity is plotted function of the doseof virus. A plateau is reached for the TRIP-(HR-GFP) vector at dose of100 ng P24/ml whereas fluorescence intensity in transduced cellsincreases with the dose of TRIP+vector. This could reflect thelimitation in nuclear import of the pre-integrative forms of theTRIP-vector and the increasing number of integrated copies per cellafter transduction with increasing doses of the TRIP-GFP vector. Thethird plot integrates both aspects and shows the resulting effect of theDNA triplex sequence on GFP fluorescence activity in human HSC. Asshown, the presence of the DNA triplex sequence in the HIV vector inducean increased GFP production in HSC by a factor of more than a ten fold.

Example 12 A Fraction of Integrated Copies of HIV Vector Genomes RemainsSilent in Human Hematopoietic Transduced Cells

It is possible that inactivation of the integrated transgene can occur.The transfer efficiency was always better when it is evaluated by thepercentage of transduced progenitor-derived cell colonies determined byPCR assay for the integrated lentiviral vector rather than when it isevaluated by the percentage of CD34+/eGFP+ cells determined by FACSanalysis 48 hours after the end of the transduction protocol. Thisreflects the occurrence of transcriptionally inactive proviruses eitherdue to their integration site or to the progressive and randominactivation of the provirus while the cells proliferate anddifferentiate. We have observed colonies derived from a single myeloidclonogenic progenitor that some of the subclones could be GFP brightwhereas others were negative, reflecting the random transcriptionalinactivation of the integrated transgene in this phenotypicallyhomogenous clonal progeny.

Example 13 HIV Vector Containing a Deleted Version of the U3 Region ofthe LTR and an Internal EFIα are More Potent Systems for theTransduction of Hematopoietic Stem Cells

In FIG. 9, we compared the ability of various HIV-1 derived vectors,including the DNA triplex sequence, to transduce human cord blood CD34+cells. The effect of deletion of most of the U3 region of the 3′LTR onGFP transduction and expression in CD34+ cells was analyzed. Thecomparison of vectors containing an intact HIV-1 LTR or a U3 deletedversion was conducted in the context of a CMV internal promoter or anEFIα internal promoter (Kim et al., 1990) to drive the expression of theGFP reporter gene. All transduction experiments were conducted using thesame concentration of vector particles (500 ng P24/ml) afternormalization of vector stocks using a commercially available ELISAassay for the P24 (Capsid protein) HIV-1 antigen (Dupont). Flowcytometry analysis (FACS) were performed either at 48 hours (FIG. 9A) or120 hours (FIG. 9B) after the 24 hour transduction period.

Interestingly, deletion of the U3 region of the LTR in HIV-1 vectorsinduced a slight increase in the percentage of GFP positive cells in allcases. This increase was modest when analyzed at 48 hours. At this time,a pseudotransduction mechanism might be responsible for a fraction ofthe GFP positive cells. Pseudotransduction is the direct delivery of GFPproteins to target cells by the fusing retroviral vector particle,without the need for an actual integration of the retroviral vectorgenome. The increase in the percentage of GFP positive aftertransduction by the U3 deleted versions of the HIV-1 vectors became moreevident at 120 hours after transduction (FIG. 9B). At this time, up to atwo fold increase in the percentage of GFP positive cells is seen aftertransduction by the TRIP ΔU3-EF1α-GFP vector when compared to theresults obtained with the equivalent vector but containing an intactHIV-1 LTR.

More importantly, deletion of the U3 region of the HIV-1 LTR in theHIV-1 triplex vectors induced a better expression of the GFP reporterprotein. The mean of fluorescence intensity in transduced human HSC whenanalyzed by FACS was always superior of a three to five fold factor inthe case of the U3 deleted versions than with vectors containing anintact HIV-1 LTR. This benefit of GFP expression was observed whetherthe CMV or the EF1α promoters where used as an internal promoter in theHIV-1 vector construct. The molecular mechanism explaining this enhancedexpression of GFP proteins in transduced cells is not known. Somesequence in the HIV-1 LTR may negatively influence the expression drivenby the internal promoter. Alternatively, a basal transcription initiatedat the HIV-1 LTR may interfere with the initiation of transcription atthe internal promoter.

This study also shows that the EF1 a promoter is a better promoter inhuman HSC than the CMV promoter. In FIG. 9B, the mean of GFPfluorescence intensity is three to five times better in the case of theEF la promoter than in the case of the CMV promoter.

It will be apparent to those skilled in the art that variousmodifications and variations can be made in the practice of the presentinvention without departing from the scope or spirit of the invention.

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1-43. (canceled)
 44. A method of expressing a protein of interest in vivo comprising: a) administering a lentiviral particle encoding the protein of interest to an individual; b) transducing a cell of the individual with the lentiviral particle; and c) expressing the protein of interest encoded by the lentiviral particle in the cell of the individual, wherein the lentiviral particle comprises: at least one copy of lentiviral cPPT and CTS regions; a heterologous nucleic acid sequence encoding the protein of interest under the control of an internal promoter not naturally associated with the heterologous nucleic acid sequence; a lentiviral LTR that is deleted for the promoter and the enhancer of U3; a lentiviral integrase protein; a lentiviral reverse transcriptase protein; a lentiviral Gag protein; and an envelope protein.
 45. The method of claim 44, wherein the lentiviral cPPT and CTS regions are HIV-1, HIV-2 VISNA, EIAV, FIV, or CAEV cPPT and CTS regions.
 46. The method of claim 45, wherein the lentiviral cPPT and CTS regions are HIV-1 cPPT and CTS regions.
 47. The method of claim 45, wherein the lentiviral LTR is an HIV-1, HIV-2 VISNA, EIAV, FIV, or CAEV LTR.
 48. The method of claim 46, wherein the lentiviral LTR is an HIV-1 LTR.
 49. The method of claim 47, wherein the lentiviral integrase and reverse transcriptase proteins are HIV-1, HIV-2 VISNA, EIAV, FIV, or CAEV integrase and reverse transcriptase proteins.
 50. The method of claim 48, wherein the lentiviral integrase and reverse transcriptase proteins are HIV-1 integrase and reverse transcriptase proteins.
 51. The method of claim 49, wherein the lentiviral Gag protein is an HIV-1, HIV-2 VISNA, EIAV, FIV, or CAEV Gag protein.
 52. The method of claim 50, wherein the lentiviral Gag protein is an HIV-1 Gag protein.
 53. The method of claim 44, wherein the envelope protein is a VSV envelope protein.
 54. A lentiviral particle comprising: a) at least one copy of lentiviral cPPT and CTS regions; b) a heterologous nucleic acid sequence encoding a protein of interest under the control of an internal promoter not naturally associated with the heterologous nucleic acid sequence; c) a lentiviral LTR that is deleted for the promoter and the enhancer of U3: d) a lentiviral integrase protein; e) a lentiviral reverse transcriptase protein; f) a lentiviral Gag protein; and g) an envelope protein.
 55. A method of introducing a heterologous nucleic acid sequence into the nucleus of a cell comprising transducing the cell with a lentiviral vector particle comprising a nucleic acid comprising: (a) at least one copy of the cPPT and CTS cis-acting regions of a retrovirus, wherein said cPPT and CTS regions induce a three-stranded DNA structure, (b) a heterologous nucleic acid sequence, and (c) an HIV-1 LTR that is deleted for the promoter and the enhancer of U3.
 56. A method for producing a lentiviral gene transfer vector that promotes nuclear import of a gene of interest comprising generating a gene transfer vector comprising: (a) at least one copy of the cPPT and CTS cis-acting regions of a retrovirus, wherein said cPPT and CTS regions induce a three-stranded DNA structure, (b) a gene of interest, (c) an HIV-1 LTR that is deleted for the promoter and the enhancer of U3, and (d) a cis-acting lentiviral encapsidation sequence.
 57. An isolated HIV-1 expression vector comprising: (a) an isolated or purified nucleic acid comprising one copy of the cPPT and CTS cis-acting regions of HIV-1, wherein said cPPT and CTS regions induce a three-stranded DNA structure, (b) a heterologous nucleic acid sequence, and (c) an HIV-1 LTR that is deleted for the promoter and the enhancer of U3.
 58. An isolated nucleic acid comprising the nucleic acid sequence of the EcoRI/Mlul insert of vector pTRIPΔU3 EF1αGFP in which the promoter and enhancer of the U3 sequence have been deleted, deposited at National Collection of Cultures of Microorganismes, Accession Number I-2328.
 59. An isolated HIV-1 expression vector comprising: (a) an isolated or purified nucleic acid comprising at least one copy of the cPPT and CTS cis-acting regions of a retrovirus, wherein said cPPT and CTS regions induce a three-stranded DNA structure, (b) a heterologous nucleic acid sequence, and (c) an HIV-1 LTR that is deleted for the promoter and the enhancer of U3.
 60. A process for expressing at least one heterologous nucleic acid of interest, in vitro, wherein the process comprises: (a) exposing a target cell the expression vector of claim 59 under conditions that permit uptake of the expression vector into the target cell to create a recombinant cell and (b) culturing the recombinant cell under conditions that permit the heterologous nucleic acid sequence to be transferred to the nucleus of the recombinant cell and permit the heterologous nucleic acid sequence to be expressed.
 61. An isolated recombinant cell transduced with a lentiviral vector particle comprising a nucleic acid comprising: (a) at least one copy of the cPPT and CTS cis-acting regions of a lentivirus, wherein said cPPT and CTS regions induce a three-stranded DNA structure, (b) a heterologous nucleic acid sequence, and (c) an HIV-1 LTR that is deleted for the promoter and the enhancer of U3.
 62. A method for producing an isolated recombinant cell comprising the transduction of a cell with a lentiviral vector particle, wherein the lentiviral vector particle comprises a nucleic acid comprising: (a) at least one copy of the cPPT and CTS cis-acting regions of a lentivirus, wherein said cPPT and CTS regions induce a three-stranded DNA structure, (b) a heterologous nucleic acid sequence, and (c) an HIV-1 LTR that is deleted for the promoter and the enhancer of U3.
 63. A recombinant lentiviral vector particle comprising Gag, Pol, and Env proteins and a replication-deficient lentiviral vector comprising: (a) at least one copy of the cPPT and CTS cis-acting regions of a retrovirus, wherein said cPPT and CTS regions induce a three-stranded DNA structure upon reverse transcription, (b) a transgene of interest, and (c) a deletion of the promoter and the enhancer sequences of U3 region of the HIV-1 LTR. 